1. Field of the Invention
The present invention relates to a method for introducing a foreign matter into a living cell. Particularly, the invention relates to such a foreign matter into a living cell with use of a laser.
2. Related Art Statement
As the methods for introducing foreign matters such as genetic substances into living cells, there are known a method for directly introducing a foreign matter into a living cell with use of a fine glass tube (microinjection method), a method for applying a cell-fusing technique (a liposome fusion method and a protoplast fusion method), an introducing method using an infecting process (Agrobacterium method), a membrane permeability-promoting method (electropolation method), etc. when broadly classified.
According to the microinjection method, a fine micropipette is prepared with use of a glass tube, connecting it with a micromanipulator, and introducing a liquid of a foreign matter into a cell one by one under observation with a microscope. This method is a relatively excellent foreign matter-introducing method for animal cells having no cell walls.
As the cell fusion technique-applying methods, there are the liposome method, the protoplast fusion method, etc. According to the protoplast fusion method, a colon bacterium (Escherichia coli) or the like having a coloned plasmide is converted to a protoplast, which is fused with a target cell with ethylene glycol or the like, thereby introducing a gene into the cell. According to the liposome fusion method, a foreign matter is introduced into a target cell by using a liposome as a carrier.
As the infection process-utilizing introduction method, there is known a method for introducing a gene by using an Agrobacterium carrying a Ti plasmid.
According to the electropolation method based on the promotion of the membrane permeability, a cell is suspended in a solution of a foreign matter, and DC pulses at a high voltage upon the resultant solution or suspension, and thereby the foreign matter is introduced into the cell.
There is also known a gene gun method in which a fine metal particle coated with DNA is thrown into a cell of an organism, and thereby a gene is introduced into the cell through a cell wall and/or cell membrane. Furthermore, there is recently known a method for introducing a foreign matter into a cell by using a laser. These conventional methods for introducing the foreign matters into the cells are used mainly for transforming the target cells through the introduction of the foreign genes.
However, with recent progresses in the biotechnology, there have been the possibilities in diagnosis and treatment of diseases, molecular breeding of agricultural products imparted with various tolerances, production of useful products including livestock, etc. through introducing, into living cells, foreign matters other than genes, e.g., organella such as chloroplasts, nuclei, chromosomes and mitochondoria, physiologically active materials and indicators, functional proteins. Further, production of directly transformed bodies having undergone de-differentiation nor re-differentiation is conceivable in the case of plant, for example, by each introducing a foreign matter in a specified one cell of a living tissue. Thus, molecular breeding of many useful plants of which dedifferentiation or re-differentiation system has not been established are expected. Production of transformed products in which a plurality of characters are simultaneously transformed by simultaneously introducing plural kinds of foreign matters into a cell or cells is expected as well.
From this point of view, there are problems of difficulties in introducing foreign matters other than genes in the case of a gene-introducing method utilizing agrobacteria carrying Ti plasmides, a protoplast fusion method and a gene gun method among the conventional foreign matter-introducing methods. Further, a microinjection method, a liposome method and an electropolation method have a possibility of introducing foreign matters other than genes into cells, but the liposome method and the electropolation method require protoplast conversion when applied to plant cells, so that these methods have a problem in that there is difficulty in introducing a foreign matter into a specified one cell of a specific tissue. The microinjection method has a problem in that there is extremely difficult because skill is needed to handle a plant cell having a cell wall tougher than that of an animal cell. On the other hand, a laser method can introduce a foreign matter into a given target cell, but the conventional laser method has a problem in that there is difficulty in stably introducing foreign matters having extremely small physical strength, such as artificial chromosomes, or macrostructural bodies such as organella into cells.
Therefore, there have been strongly desires for development of a method which is easy to manipulate and has general-purpose applicability, which transforms a specified one cell as a target cell, and which can introduce a foreign matter such as not only genes but also foreign matters having extremely small physical strength, such as artificial chromosomes, or foreign matters such as macrostructural bodies such as organella into living cells irrespective of species of organisms. However, such a method for introducing the foreign matter into the living cell has not been known yet.